The regulatory loci and the effectors that modulate the rate fatty acid Beta-oxidation in liver and heart mitochondria are known. A knowledge of metabolic regulation of Veta-oxidation is essential to understanding the factors that control fatty acid catabolism especially in diseases, such as diabetes, that perturb fatty acid oxidation in liver and heart. We propose to study the acyl CoA dehydrogenases, soluble flavoprotein oxidoreductases, that catalyze the desaturation of acyl CoA thiolesters in each cycle of Veta-oxidation. We will also study the electron transfer flavoprotein (ETF) and ETF dehydrogenase which catalyze the transfer of electrons from the primary dehydrogenases to the mitochondrial electron transport system. Previous studies with whole mitochondria indicate that the reactions catalyzed by the dehydrogenases are rat-limiting in Beta-oxidation; moreover, the equilibria for reduction of the primary dehydrogenases by optimal chain length substrates strongly favors dehydrogenase reduction. These two observations suggest that the flavoprotein dehydrogenases should be subject to metabolic regulation. Little is known about the chemistry of the redox sequence catalyzed by the three proteins and metabolic control of this redox sequence has not been investigated. Recent advances in our laboratory permit purification of an acyl CoA dehydrogenase and ETF in quantities sufficient for mechanistic, redox and regulatory studies. Using steady-state and transient kinetic techniques, electrochemical and electron spin resonance methods we will elucidate the basic chemical and mechanistic properties of the proteins that catalyze the redox sequence. From a konwledge of basic properties of the activities of these proteins, alone and acting in sequence, we will be able to determine: (1) Whether this redox sequence is metabolically controlled, (2) The mode of control, (3) The physiological significance of the control, and (4) The kinetic relationships between the dehydrogenase reaction and other steps in the Beta-oxidation cycle.